New FK-506 cytosolic binding protein

(19)

(11)

EP 0 379 342 A3

(12)	EUROPEAN PATENT APPLICATION

(88)	Date of publication A3:
	28.11.1990 Bulletin 1990/48

(43)	Date of publication A2:
	25.07.1990 Bulletin 1990/30

(21)	Application number: 90300459.6

(22)	Date of filing: 17.01.1990

(51)	International Patent Classification (IPC)⁵: C07K 15/12, G01N 33/68

(84)	Designated Contracting States:
	CH DE FR GB IT LI NL

(30)

Priority:

19.01.1989 US 300659
21.06.1989 US 369576

(71)	Applicant: Merck & Co., Inc.
	Rahway New Jersey 07065-0900 (US)

(72)	Inventors:
	Siekierka, John J. Nutley, NJ 07110 (US) Staruch, Marie J. Toms River, NJ 08753 (US) Sigal, Nolan H. Westfield, NJ 07090 (US) Mumford, Richard A. Red Bank, NJ 07701 (US) Hung, Hsueh-Yun East Brunswick, NJ 08816 (US)

(74)	Representative: Cole, William Gwyn et al
	European Patent Department Merck & Co., Inc. Terlings Park Eastwick Road Harlow Essex CM20 2QR Harlow Essex CM20 2QR (GB)

(56)

References cited: :

(54)	New FK-506 cytosolic binding protein

(57) A new homogeneous cytosolic binding (HCB) protein, having a specific binding activity of about 26 µg FK-506 per mg protein and a molecular weight of about 10-12 kilodaltons, reversibly binds the immunosuppressant FK-506 but not cyclosporine A (CSA). The protein is stable to heating at 56 degrees C. for 30 minutes retaining its FK-506 binding affinity, and has the (partial) amino terminal amino acid sequence: H₂N-Gly-Val-Gln-Val-Glu-Thr-Ile-Ser-Pro-Gly-Asp-Gly-Arg-Thr-Phe-Pro-Lys-Arg-Gly-Gln-Thr-X-Val-Val-His-Tyr-Thr-Gly-Met-Leu-Glu-Asp-Gly-Lys-Lys-Phe-Asp (wherein X is undefined). The HCB protein is isolated from the cytosol of mammalian tissues, preferably human neoplastic T-cell lines, e.g. Jurkat, and can be used in diagnostic and purification procedures involving FK-506 macrolide type immunosuppressants. The HCB protein also catalyses the cis-trans isomerization of proline-containing peptide bonds.

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